Due to their chemical structure, glucosaminoglucans exhibit polydispersity of molecular weight and the correlation between biological activity, structure and weight has not yet been clarified. Recently, several reports have appeared in the literature showing that fractionation of glucosaminoglucans on the basis of the molecular weight or on the basis of their affinity for other substances having appropriate biological activity is possible. Certainly, it would be very desirable from a therapeutical point of view to obtain purer fractions and fractions with a higher degree of specific activity, whether it is anticoagulant, antilipemic or antithrombotic activity. For instance, in the case of heparin which exhibits an average distribution of molecular weight between 5000 and 20000 it has been reported by L. O. Andersson, T. W. Barrowcliffe, E. Holmer, E. A. Johnson and G. E. C. Sims, Thrombosis Research, 9, 575-583, (1976); R. Jordan, D. Beeler, R. Rosenberg, J. Biol. Chem., 254, 2902-2913 (1979); H. J. Rodriguez and A. J. Vanderwielen, J. Pharm. Sci., 68, 588-591 (1979), that fractions of average molecular weight between 5000 and 14,000 exhibit an anticoagulant specific activity in the range of 40% of a fraction having an average molecular weight of 12000.
The fractions of molecular weight 5000, which are obtained by chromatography and affinity towards antithrombin, give fractions some of which exhibit a high anticoagulant activity, for instance 250-300 U.I./mg, whereas other fractions exhibit antithrombotic or antilipemic activity. These methods of fractionation, in general, have been carried out on a laboratory scale using chromotographic techniques. Even when it is feasible to extend these methods to a larger scale, it is likely that the fractions become contaminated, and that the stability of the chromotographic supports is affected, both phenomena seriously interfering with the application of these methods to an industrial scale as continuous processes. Further, in addition to the problems, relating to enrichment or isolation of activities, there are other problems inherent to commercial glucosaminoglucans. More specifically, glucosaminoglucans are manufactured by organs of several animals, such as bovine, swine, etc., such as lungs, intestinal mucosa, etc. The extraction processes are very complex because they include the step of enzymatic proteolysis of the glycoproteins starting material, salt formation with ammoniacal quaternary salts or the use of ion exchange resins and, in general, more than one precipitation with alcoholic solvents in the presence of salts, etc., is required. The crude product obtained from the first purification must then be subjected to time-consuming purifications which lead to various commercial glucosaminoglucans of high purity. In any event, in the purified product, it is likely that there remains:
(a) organic or inorganic impurities which on one hand do not exhibit specific biological activities and, on the other hand, are likely to cause serious technological problems during actual use, and further they are likely to lower the specific biological activity, for instance the U. I. anticoagulant/mg, (b) impurities, the presence of which is not acceptable because of an inherent negative potential activity on a biological level. Typical among these types of impurities are the oxidizing substances which are utilized in the final step to decolorize the product.
Several methods have been proposed, some of which have not been patented, which lead more or less to the elimination of these impurities. These methods are based on the use of ion exchange resins, selective precipitation, etc.